RNA polymerase is the central enzyme of gene expression in all domains of life. Changes in gene expression patterns punctuate the progress of normal and neoplastic development of eukaryotes and onset of virulence in bacteria. One of the most exciting goals in transcription studies now is identification of the 'hot spots' on RNA polymerase that mediate its interactions with regulators; these sites could serve as targets for design of the bacterial RNA polymerase inhibitors. Recent structural studies of large, ~450 kDa multi-subunit bacterial RNA polymerases revealed a highly intricate architecture of the enzyme. Various cavities and channels on the enzyme surface that provide access to the functionally important protein regions and to the nucleic acid chains likely serve as targets for the regulatory transcription factors. Among these targets, the RNA polymerase substrate entry (secondary) channel has been recently recognized as a binding site for an ever-growing number of transcription regulators. Determinants in the secondary channel mediate stringent response of bacteria to stress and starvation, provide tight homeostatic control of the rRNA expression, and underlie the 3 known activities of RNA polymerase: nucleotide addition, endo- and exo-nucleolytic cleavages of the nascent RNA, and in turn likely control the fidelity of transcription. The goal of this project is to structurally and functionally characterize the secondary channel as one of the crucial regulatory sites in RNA polymerase to aid in understanding of gene regulation and design of novel antibacterial compounds.
Specific aims of this study will be as follows. First, to elucidate the structural basis of the mechanism by which transcript cleavage factors 'rescue' arrested transcription complexes. Structures of RNA polymerase elongation complexes with bound transcript cleavage factors GreA, GreAl, GreA2 and GreB will be determined at the atomic level using the nucleic acid scaffolds that mimic the backtracked elongation complexes, their native substrates. Second, to provide further insights into regulation of the rRNA synthesis and the molecular mechanism of stringent response, structure of the RNA polymerase with bound major regulator, alarmone ppGpp, and its protein co-regulator DksA, will be determined. Third, a combination of biochemical and genetic approaches (structure-guided mutagenesis, in vitro transcription assays, crosslinking, etc) will be used to dissect the determinants in RNA polymerase, nucleic acids, and transcription factors that mediate control of transcription through the secondary channel.
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